JP2013252077A - Method for purifying vegetable oil and method for producing lysophospholipid from vegetable oil - Google Patents
Method for purifying vegetable oil and method for producing lysophospholipid from vegetable oil Download PDFInfo
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- JP2013252077A JP2013252077A JP2012128617A JP2012128617A JP2013252077A JP 2013252077 A JP2013252077 A JP 2013252077A JP 2012128617 A JP2012128617 A JP 2012128617A JP 2012128617 A JP2012128617 A JP 2012128617A JP 2013252077 A JP2013252077 A JP 2013252077A
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- 235000015112 vegetable and seed oil Nutrition 0.000 title claims abstract description 58
- 239000008158 vegetable oil Substances 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 130
- 102000004190 Enzymes Human genes 0.000 claims abstract description 97
- 108090000790 Enzymes Proteins 0.000 claims abstract description 97
- 108010058864 Phospholipases A2 Proteins 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 claims abstract description 34
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims abstract description 25
- 102000015439 Phospholipases Human genes 0.000 claims abstract description 25
- 108010064785 Phospholipases Proteins 0.000 claims abstract description 25
- 235000019626 lipase activity Nutrition 0.000 claims abstract description 25
- 108020002496 Lysophospholipase Proteins 0.000 claims abstract description 20
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- 102100037611 Lysophospholipase Human genes 0.000 claims abstract 13
- 239000003921 oil Substances 0.000 claims description 44
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
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- 229960000318 kanamycin Drugs 0.000 description 5
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
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- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 2
- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 description 2
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
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- 125000002252 acyl group Chemical group 0.000 description 2
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- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 description 2
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- 238000004945 emulsification Methods 0.000 description 2
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- 150000002327 glycerophospholipids Chemical group 0.000 description 2
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- 239000002904 solvent Substances 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 235000019483 Peanut oil Nutrition 0.000 description 1
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- 235000019485 Safflower oil Nutrition 0.000 description 1
- 101100397226 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp4 gene Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000315804 Streptomyces avermitilis MA-4680 = NBRC 14893 Species 0.000 description 1
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
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- 235000005713 safflower oil Nutrition 0.000 description 1
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- ROKRAUFZFDQWLE-UHFFFAOYSA-M sodium;1-ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylate Chemical compound [Na+].C1=C(C)N=C2N(CC)C=C(C([O-])=O)C(=O)C2=C1 ROKRAUFZFDQWLE-UHFFFAOYSA-M 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、植物油を精製する方法および植物油からリゾリン脂質を製造する方法に関する。 The present invention relates to a method for purifying vegetable oil and a method for producing lysophospholipid from vegetable oil.
油原料植物から圧搾した油、または抽出し、溶剤を除去した油(本明細書中では、これらをまとめて「粗油」という)にはリン脂質、脂肪酸、色素、臭気化合物のような不純物が含まれるので、植物油の製造方法には、これらの不純物を除去する精製工程がある。精製工程では、粗油からリン脂質を水和析出させてガム質として取り除く(「脱ガム」)。脱ガム油は、その後、脱色(色素を活性白土などに吸着させて除去する)および脱臭(臭気化合物を蒸留除去する)に供し得る。精製工程には、化学的精製と物理的精製とがある。 Oils squeezed from oil-source plants or oils that have been extracted and solvent removed (collectively referred to herein as “crude oils”) contain impurities such as phospholipids, fatty acids, pigments, and odorous compounds. As such, the method for producing vegetable oil includes a purification step for removing these impurities. In the refining process, phospholipids are hydrated and precipitated from the crude oil as a gum (“degumming”). The degummed oil can then be subjected to decolorization (the dye is adsorbed on activated clay and removed) and deodorization (the odorous compound is distilled off). The purification process includes chemical purification and physical purification.
化学的精製においては、粗油を水で処理し(「水脱ガム」)、次いでアルカリで処理してガム質と遊離脂肪酸とに分解する(「脱酸」)。アルカリは、油および水中に存在する遊離脂肪酸と反応して石鹸を生成し、中和油を吸蔵する大量のエマルジョンを形成し、結果として油の大きな損失を招く。このプロセスにより生成される石鹸ストックは、重大な廃棄問題を引き起こす。このプロセスでは大量の水を必要とし、排水および廃棄物が多い。 In chemical refining, the crude oil is treated with water (“water degumming”) and then with alkali to break it into gums and free fatty acids (“deoxidation”). Alkali reacts with the free fatty acids present in the oil and water to produce soap, forming a large amount of emulsion that occludes the neutralized oil, resulting in a significant loss of oil. The soap stock produced by this process causes significant disposal problems. This process requires a large amount of water and is wasteful and wasteful.
物理的精製は、脱ガム油から水蒸気ストリッピングにより脱臭と共に遊離脂肪酸を取り除く処理を含む。結果として、油の損失は小さく、遊離脂肪酸は蒸留により取り除かれ、最終的に得られる製品の品質が改良される。しかし、ガム質が脱色工程前に完全に取り除かれないと、最終製品の品質が低下し得る。したがって、脱色工程前のガム質除去の処理が重要であり、このような処理として、酵素による処理(「酵素脱ガム」)が挙げられる。 Physical refining involves the removal of free fatty acids along with deodorization from the degummed oil by steam stripping. As a result, oil loss is small and free fatty acids are removed by distillation, improving the quality of the final product. However, if the gum is not completely removed before the decolorization process, the quality of the final product can be degraded. Accordingly, the gum removal treatment prior to the decolorization step is important, and examples of such treatment include treatment with enzymes (“enzymatic degumming”).
例えば、粗油または水脱ガム後の油を、ホスホリパーゼA活性を有する酵素で処理する方法(特許文献1)、水脱ガム後の油をホスホリパーゼA1、A2またはBで処理する方法(特許文献2)、および未精製大豆油をブタ膵臓由来ホスホリパーゼA2で処理する方法(特許文献3)が報告されている。また、酵素脱ガムに用いられる酵素として、Novozymes社製のLecitase 10L(ブタ膵臓由来ホスホリパーゼA2)、Lecitase Novo(フサリウム・オキシスポリウム(Fusarium oxysporium)由来ホスホリパーゼA1)、およびLecitase Ultra(サーモマイセス・ラヌギノスス(Thermomyces lanuginosus)/フサリウム・オキシスポリウム由来ホスホリパーゼA1)が知られている(非特許文献1)。ブタ膵臓由来ホスホリパーゼA2については、大量生産が難しく工業的に利用し難い、ブタ由来物を忌避する宗教の国では使用できないなどの問題がある。 For example, a method of treating crude oil or oil after water degumming with an enzyme having phospholipase A activity (Patent Document 1), and a method of treating oil after water degumming with phospholipase A1, A2 or B (Patent Document 2). ), And a method of treating unrefined soybean oil with porcine pancreatic phospholipase A2 (Patent Document 3). As enzymes used for enzyme degumming, Lecitase 10L (phospholipase A2 derived from porcine pancreas), Lecitase Novo (phosphorusase A1 derived from Fusarium oxysporium), and Lecitase Ultra (Thermomyces lanuginosus (manufactured by Novozymes) Thermomyces lanuginosus) / Fusarium oxysporum-derived phospholipase A1) is known (Non-patent Document 1). The porcine pancreas-derived phospholipase A2 has problems that mass production is difficult and industrially difficult to use, and it cannot be used in a religious country that avoids porcine origin.
ところで、リゾリン脂質は、リン脂質のアシル基におけるエステル結合1個所が加水分解され、OH基になったものである。リゾリン脂質は、通常のリン脂質より高い界面活性を有するため、食品の乳化安定性、食感、弾力性などをリン脂質以上に改善でき、他の乳化剤の使用量を節約することができる。また、食品用途の他に、化粧品用乳化剤としても利用価値がある。 By the way, the lysophospholipid is one in which one ester bond in the acyl group of the phospholipid is hydrolyzed to an OH group. Since lysophospholipids have higher surface activity than ordinary phospholipids, the emulsification stability, texture, elasticity and the like of foods can be improved over phospholipids, and the amount of other emulsifiers used can be saved. In addition to food applications, it is also useful as a cosmetic emulsifier.
このようなリゾリン脂質の調製法として、リン脂質にホスホリパーゼA2を作用させる方法が知られている。ホスホリパーゼA2はリン脂質の2−アシル基に作用し、その結果、1−アシルリゾリン脂質を生じる。 As a method for preparing such lysophospholipid, a method in which phospholipase A2 is allowed to act on phospholipid is known. Phospholipase A2 acts on the 2-acyl group of phospholipids, resulting in 1-acyl lysophospholipids.
また、粗油または粗油に水を加えてガム質を除去した脱ガム油をホスホリパーゼA活性を有する酵素で処理して水和ガム質を得ることにより、リゾレシチンを製造する方法が報告されている(特許文献4)。 In addition, a method for producing lysolecithin by treating a crude oil or a degummed oil obtained by adding water to a crude oil to remove gum is obtained by treating with an enzyme having phospholipase A activity to obtain a hydrated gum. (Patent Document 4).
本発明は、植物油を効率的に精製できる方法、およびリゾリン脂質を効率的に製造できる方法を提供することを目的とする。 An object of this invention is to provide the method which can refine | purify a vegetable oil efficiently, and the method which can manufacture a lysophospholipid efficiently.
本発明は、植物油を精製する方法を提供し、この方法は、
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、および
該処理後の植物油から脱ガム油を分離回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素は、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である。
The present invention provides a method for refining vegetable oil, the method comprising:
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity, and separating and recovering degummed oil from the treated vegetable oil,
The enzyme having phospholipase A2 activity is:
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
本発明はまた、植物油からリゾリン脂質を製造する方法を提供し、この方法は、
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、および
該処理後の植物油からリゾリン脂質を分離回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素は、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である。
The present invention also provides a method for producing lysophospholipid from vegetable oil,
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity, and separating and recovering lysophospholipids from the treated vegetable oil,
The enzyme having phospholipase A2 activity is:
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
本発明はさらに、植物油を精製し、かつ該植物油からリゾリン脂質を製造する方法を提供し、この方法は、
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、
該処理後の植物油を油相および水相に分離する工程、
該油相を回収する工程、および
該水相からリゾリン脂質を回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素は、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である。
The present invention further provides a method for purifying vegetable oil and producing lysophospholipids from the vegetable oil, the method comprising:
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity;
Separating the treated vegetable oil into an oil phase and an aqueous phase;
Recovering the oil phase, and recovering lysophospholipid from the aqueous phase,
The enzyme having phospholipase A2 activity is:
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
1つの実施形態では、上記ホスホリパーゼA2活性を有する酵素は、放線菌由来酵素である。 In one embodiment, the enzyme having phospholipase A2 activity is an actinomycete-derived enzyme.
本発明によれば、植物油を効率的に精製できる方法、およびリゾリン脂質を効率的に製造できる方法が提供される。さらに、植物油を精製すると共に、リゾリン脂質もまた製造できる方法が提供される。本発明によれば、特にブタ由来酵素を使用することなく、高いトリグリセリド収率で効率的に植物油を精製することができ、さらに植物油精製過程で発生するガム質から1−アシルリゾリン脂質を多く含むリゾリン脂質を効率的に製造できる。 According to the present invention, there are provided a method capable of efficiently purifying vegetable oil and a method capable of efficiently producing lysophospholipid. In addition, a method is provided that can purify vegetable oils and also produce lysophospholipids. According to the present invention, vegetable oil can be efficiently purified with a high triglyceride yield without using a porcine-derived enzyme, and lysolin containing a large amount of 1-acyl lysophospholipid from gums generated in the vegetable oil purification process. Lipids can be produced efficiently.
本発明の植物油を精製する方法および植物油からリゾリン脂質を製造する方法ともに、植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程を含む。 Both the method for purifying the vegetable oil of the present invention and the method for producing lysophospholipid from the vegetable oil include a step of treating the vegetable oil with water and an enzyme having phospholipase A2 activity.
本明細書中では、ホスホリパーゼA活性、リゾホスホリパーゼ活性およびリパーゼ活性は、以下のように決定される:
ホスホリパーゼA活性:大豆レシチンを基質にpH8.0、37℃で反応した際に、1分間に1μmolの脂肪酸を遊離する活性を1ユニットとする;
リゾホスホリパーゼ活性:リゾホスファチジルコリン(Sigma, L0906)を基質にpH8.0、37℃で反応した際に、1分間に1μmolの脂肪酸を遊離する活性を1ユニットとする;
リパーゼ活性:リパーゼキットS(DSファーマバイオメディカル株式会社)を使用して、以下の手順で測定する。5,5’-ジチオビス(2-ニトロ安息香酸)を含む発色液1mL、検体50μLを混和後、フェニルメチルスルホニルフルオリドを含むエステラーゼ阻害液20μLを添加する。混和後、恒温槽で30℃にて5分間予熱する。予熱終了後、三酪酸ジメルカプロール、ドデシル硫酸ナトリウムを含む基質液100μLを加え、混和後30℃にて30分間インキュベーションする。反応停止液2mLを加え、反応を停止させる。吸光度412nmを測定する。
As used herein, phospholipase A activity, lysophospholipase activity and lipase activity are determined as follows:
Phospholipase A activity: When reacting soy lecithin with a substrate at pH 8.0 and 37 ° C., the activity of releasing 1 μmol of fatty acid per minute is defined as 1 unit;
Lysophospholipase activity: When lysophosphatidylcholine (Sigma, L0906) is reacted with a substrate at pH 8.0 and 37 ° C., the activity of releasing 1 μmol of fatty acid per minute is defined as 1 unit;
Lipase activity: Measured by the following procedure using Lipase Kit S (DS Pharma Biomedical Co., Ltd.). Mix 1 mL of color developing solution containing 5,5'-dithiobis (2-nitrobenzoic acid) and 50 μL of sample, and then add 20 μL of esterase inhibitor containing phenylmethylsulfonyl fluoride. After mixing, preheat for 5 minutes at 30 ° C. in a thermostatic bath. After preheating is completed, 100 μL of a substrate solution containing dimercaprol tributyrate and sodium dodecyl sulfate is added, and the mixture is incubated at 30 ° C. for 30 minutes. Add 2 mL of stop solution to stop the reaction. The absorbance is measured at 412 nm.
「ホスホリパーゼA2活性を有する酵素」は、ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下、好ましくは1%以下、より好ましくは0.5%以下である酵素である。「ホスホリパーゼA2」(以下、「PLA2」ともいう)は、水の存在下、グリセロリン脂質(例えば、レシチン)の2位のエステル結合を加水分解し、1−アシル−2−リゾリン脂質(以下、単に「1−アシルリゾリン脂質」ともいう:例えば、1−アシルリゾレシチン)と脂肪酸とを生成する触媒作用を有する酵素をいう。「ホスホリパーゼA1」(以下、「PLA1」ともいう)は、水の存在下、グリセロリン脂質(例えば、レシチン)の1位のエステル結合を加水分解し、2−アシル−1−リゾリン脂質(以下、単に「2−アシルリゾリン脂質」ともいう:例えば、2−アシルリゾレシチン)と脂肪酸とを生成する触媒作用を有する酵素をいう。2−アシルリゾリン脂質は次いで1−アシルリゾリン脂質に転位し得、ホスホリパーゼA1は、この1−アシルリゾリン脂質をさらに分解し得る。各酵素のPLA1活性またはPLA2活性は、公知の方法に従って測定し得、PLA2活性に対するPLA1活性は、得られた活性測定値に基づいて算出し得る。酵素のPLA2活性に対するPLA1活性は、10%レシチン溶液(Ultralec P(ADM社)を水に分散)0.2mLに、酵素を300U/gとなるように添加し、37℃にて60分反応させた後、クロロホルム:メタノール(2:1(v/v))10mLを添加して、脂質を抽出し、遠心した上清を高速液体クロマトグラフ(HPLC)分析(例えば、非特許文献2に記載のHPLC法に従う。溶離液はエタノール(95%(v/v)):20mMシュウ酸=92:8(v/v)を使用)に供し、1−アシルリゾリン脂質および2−アシルリゾリン脂質のピーク面積を求め、それぞれのリゾリン脂質を定量し、これらのそれぞれの定量値からPLA1活性とPLA2活性との比を算出することにより決定し得る。「ホスホリパーゼA2活性に対するホスホリパーゼA1活性」は、PLA2活性を100としたときのPLA1活性を%として表す。 The “enzyme having phospholipase A2 activity” is an enzyme having a phospholipase A1 activity with respect to the phospholipase A2 activity of 5% or less, preferably 1% or less, more preferably 0.5% or less. “Phospholipase A2” (hereinafter also referred to as “PLA2”) hydrolyzes the ester bond at the 2-position of glycerophospholipid (for example, lecithin) in the presence of water, and 1-acyl-2-lysophospholipid (hereinafter simply referred to as “phospholipase A2”). Also referred to as “1-acyl lysophospholipid”: for example, an enzyme having a catalytic action to produce 1-acyl lysolecithin) and a fatty acid. “Phospholipase A1” (hereinafter also referred to as “PLA1”) hydrolyzes the ester bond at the 1-position of glycerophospholipid (for example, lecithin) in the presence of water, to give 2-acyl-1-lysophospholipid (hereinafter simply referred to as “phospholipase A1”). Also referred to as “2-acyl lysophospholipid”: for example, an enzyme having a catalytic action to produce 2-acyl lysolecithin) and a fatty acid. The 2-acyl lysophospholipid can then be translocated to the 1-acyl lysophospholipid, and phospholipase A1 can further degrade the 1-acyl lysophospholipid. PLA1 activity or PLA2 activity of each enzyme can be measured according to a known method, and PLA1 activity relative to PLA2 activity can be calculated based on the obtained activity measurement value. The PLA1 activity relative to the PLA2 activity of the enzyme was added to 0.2 mL of a 10% lecithin solution (Ultralec P (ADM) dispersed in water) at 300 U / g and reacted at 37 ° C. for 60 minutes. Thereafter, 10 mL of chloroform: methanol (2: 1 (v / v)) was added to extract the lipid, and the centrifuged supernatant was subjected to high performance liquid chromatographic (HPLC) analysis (for example, HPLC described in Non-Patent Document 2). The eluent is subjected to ethanol (95% (v / v)): 20 mM oxalic acid = 92: 8 (v / v)), and the peak areas of 1-acyl lysophospholipid and 2-acyl lysophospholipid are determined, Each lysophospholipid can be quantified and determined by calculating the ratio of PLA1 activity to PLA2 activity from each quantified value. “Phospholipase A1 activity relative to phospholipase A2 activity” represents PLA1 activity as% when PLA2 activity is taken as 100.
本発明の方法で使用される酵素は、ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である。該酵素は、ホスホリパーゼA活性に対するリゾホスホリパーゼ活性が5%以下、好ましくは1%以下、より好ましくは0.5%以下である。該酵素は、ホスホリパーゼA活性に対するリパーゼ活性が2%以下、好ましくは1%以下、より好ましくは0.5%以下、さらにより好ましくは0.3%以下である。ホスホリパーゼA活性に対するリゾホスホリパーゼ活性(%)およびホスホリパーゼA活性に対するリパーゼ活性(%)は、上記の測定法による活性測定値に基づいて算出することにより決定される。 The enzyme used in the method of the present invention has a lysophospholipase activity of 5% or less and a lipase activity of 2% or less with respect to the phospholipase A activity. The enzyme has a lysophospholipase activity with respect to phospholipase A activity of 5% or less, preferably 1% or less, more preferably 0.5% or less. The enzyme has a lipase activity relative to phospholipase A activity of 2% or less, preferably 1% or less, more preferably 0.5% or less, and even more preferably 0.3% or less. The lysophospholipase activity (%) with respect to the phospholipase A activity and the lipase activity (%) with respect to the phospholipase A activity are determined by calculation based on the activity measurement value obtained by the above-described measurement method.
本発明の方法で使用される酵素は、上で説明した活性を満たす限り、酵素の起源および調製方法は問わない。酵素の起源は、特に限定されないが、一般的には微生物起源の酵素が用いられる。このような微生物は、特に限定されず、例えば、放線菌(例えば、ストレプトマイセス属(Streptomyces)、アクチノマヂューラ属(Actinomadura)、またはフランキア属(Frankia)に属する放線菌)が挙げられる。また、微生物は、天然に存在する野生型あるいは形質転換体のいずれであってもよい。酵素は、一般的には、微生物から単離または抽出された精製酵素または粗精製酵素として用いられる。酵素を固定化して用いてもよく、あるいは微生物菌体自体をそのまま用いてもよい。好ましくは、ストレプトマイセス属に属する放線菌由来酵素であり、このような酵素としては、例えば、ストレプトマイセス・ビオラセルバー(Streptomyces violaceoruber)NBRC15146株由来ホスホリパーゼA2(商品名「PLA2ナガセ」、ナガセケムテックス株式会社製)、ストレプトマイセス・エバミチルス(Streptomyces avermitilis)NBRC14893株由来ホスホリパーゼA2などが好適に用いられる。 The enzyme used in the method of the present invention may be of any origin and preparation method as long as the activity described above is satisfied. The origin of the enzyme is not particularly limited, but generally an enzyme of microbial origin is used. Such a microorganism is not particularly limited, and examples thereof include actinomycetes (for example, actinomycetes belonging to Streptomyces, Actinomadura, or Frankia). The microorganism may be a naturally occurring wild type or a transformant. The enzyme is generally used as a purified enzyme or a crudely purified enzyme isolated or extracted from a microorganism. The enzyme may be immobilized and used, or the microbial cell itself may be used as it is. Preferably, it is an actinomycete-derived enzyme belonging to the genus Streptomyces. Examples of such an enzyme include phospholipase A2 derived from Streptomyces violaceoruber NBRC15146 strain (trade names “PLA2 Nagase”, Nagase ChemteX) Co., Ltd.), Streptomyces avermitilis NBRC14893 strain-derived phospholipase A2, and the like are preferably used.
ストレプトマイセス・ビオラセルバーNBRC15146株由来ホスホリパーゼA2は、上で説明した活性を満たし、好ましくは下記の理化学的性質を有する:
(a)作用および基質特異性:1,2−ジアシルリン脂質の2位のアシル基がグリセロール骨格と結合する部位を加水分解し、2−アシル基を遊離する。
(b)至適pH:7〜9
(c)安定pH:4〜10
(d)至適温度:50℃
(e)安定温度:30−60℃
(f)分子量:SDS−PAGEによる測定で14kDaである。
The phospholipase A2 derived from Streptomyces violacervar strain NBRC15146 satisfies the activity described above and preferably has the following physicochemical properties:
(A) Action and substrate specificity: Hydrolyzes the site where the 2-position acyl group of 1,2-diacylphospholipid binds to the glycerol skeleton to release the 2-acyl group.
(B) Optimal pH: 7-9
(C) Stable pH: 4-10
(D) Optimal temperature: 50 ° C
(E) Stable temperature: 30-60 ° C
(F) Molecular weight: 14 kDa as measured by SDS-PAGE.
ストレプトマイセス・エバミチルスNBRC14893株由来ホスホリパーゼA2は、例えば、以下の調製例1に記載のようにして調製され得る。 The phospholipase A2 derived from Streptomyces ebamitilus NBRC14893 can be prepared, for example, as described in Preparation Example 1 below.
ストレプトマイセス・ビオラセルバーNBRC15146株由来ホスホリパーゼA2は、例えば配列番号2に記載のアミノ酸配列を有し、ストレプトマイセス・エバミチルスNBRC14893株由来ホスホリパーゼA2は、例えば配列番号4に記載のアミノ酸配列を有する。本酵素は、上で説明した活性を満たす限り、天然型酵素のアミノ酸配列(例えば、配列番号2または4に記載のアミノ酸配列)に対して1または数個のアミノ酸が置換、欠失、挿入、および/または付加したアミノ酸配列を有する酵素であってもよい。当業者であれば、例えば、当業者が通常用いる部位特異的変異導入法などを用いて、適宜置換、欠失、挿入、および/または付加変異を導入することにより、タンパク質の構造を改変することができる。本発明において、置換、欠失、挿入、および/または付加することができるアミノ酸残基数は、通常45以下、例えば30以下、あるいは20以下、好ましくは16以下、より好ましくは5以下、さらに好ましくは0〜3アミノ酸残基である。また、アミノ酸の変異は自然界において生じることもあるので、人工的にアミノ酸を変異した酵素のみならず、自然界においてアミノ酸が変異した酵素も、上で説明した活性を満たす限り、本発明において用いられ得る。 The phospholipase A2 derived from Streptomyces violacerbar NBRC15146 strain has the amino acid sequence described in, for example, SEQ ID NO: 2, and the phospholipase A2 derived from Streptomyces evarcillus NBRC14893 strain has, for example, the amino acid sequence described in SEQ ID NO: 4. As long as the enzyme satisfies the activity described above, one or several amino acids are substituted, deleted, inserted, or inserted into the amino acid sequence of the natural enzyme (for example, the amino acid sequence described in SEQ ID NO: 2 or 4). And / or an enzyme having an added amino acid sequence. A person skilled in the art can modify the structure of a protein by appropriately introducing substitution, deletion, insertion, and / or addition mutation using, for example, a site-specific mutagenesis method ordinarily used by those skilled in the art. Can do. In the present invention, the number of amino acid residues that can be substituted, deleted, inserted and / or added is usually 45 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably. Is 0 to 3 amino acid residues. In addition, since amino acid mutations may occur in nature, not only enzymes in which amino acids have been artificially mutated but also enzymes in which amino acids have been mutated in nature can be used in the present invention as long as the activities described above are satisfied. .
上記天然型酵素のアミノ酸配列(例えば、配列番号2または4に記載のアミノ酸配列)に対して相同性を有するアミノ酸配列を有するタンパク質も、上で説明した活性を満たす限り、本発明において用いられ得る。好ましくは、配列番号2または4に記載のアミノ酸配列と少なくとも70%、より好ましくは少なくとも80%、さらにより好ましくは少なくとも90%、なおより好ましくは少なくとも95%、さらになおより好ましくは少なくとも99%の相同性を有するアミノ酸配列を有するタンパク質であり得る。タンパク質の相同性(ホモロジー)検索は、例えばSWISS-PROT、PIR、DADなどのタンパク質のアミノ酸配列に関するデータベース、またはDDBJ、EMBL、あるいはGene-BankなどのDNA配列に関するデータベース、DNA配列を元にした推定アミノ酸配列に関するデータベースなどを対象に、BLAST、FASTAなどのプログラムを利用して、例えば、インターネットを通じて行うことができる。タンパク質の活性の確認は、上記に記載の手順を利用して行い得る。 A protein having an amino acid sequence having homology to the amino acid sequence of the natural enzyme (for example, the amino acid sequence described in SEQ ID NO: 2 or 4) can also be used in the present invention as long as the activity described above is satisfied. . Preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% with the amino acid sequence set forth in SEQ ID NO: 2 or 4. It can be a protein having an amino acid sequence with homology. Protein homology search, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, or databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and estimation based on DNA sequences For example, it can be performed through the Internet using a program such as BLAST or FASTA for a database relating to an amino acid sequence. Confirmation of the activity of the protein can be performed using the procedure described above.
本発明で用いられ得る酵素は、例えば、配列番号1または3に記載の塩基配列を含むポリヌクレオチドまたは遺伝子によってコードされ得る。配列番号1および3に記載の塩基配列を含むポリヌクレオチドまたは遺伝子はそれぞれ、配列番号2および4に記載のアミノ酸配列を含むタンパク質、すなわち、ストレプトマイセス・ビオラセルバーNBRC15146株由来ホスホリパーゼA2およびストレプトマイセス・エバミチルスNBRC14893株由来ホスホリパーゼA2をコードする。 The enzyme that can be used in the present invention can be encoded by, for example, a polynucleotide or gene containing the nucleotide sequence set forth in SEQ ID NO: 1 or 3. Polynucleotides or genes containing the nucleotide sequences set forth in SEQ ID NOs: 1 and 3, respectively, are proteins containing the amino acid sequences set forth in SEQ ID NOs: 2 and 4, ie, phospholipase A2 from Streptomyces violacerber NBRC15146 strain and Streptomyces Encodes phospholipase A2 derived from Ebamitilus NBRC14893 strain.
本発明で用いられ得る酵素はまた、例えば、配列番号1または3に記載の塩基配列を含むポリヌクレオチドに相補的な塩基配列を有するポリヌクレオチドとストリンジェントな条件でハイブリダイズできるポリヌクレオチドによってコードされ、かつ上で説明した活性を満たす酵素であり得る。ストリンジェントな条件でハイブリダイズできるポリヌクレオチドとは、配列番号1または3に記載の塩基配列中の任意の少なくとも20個、好ましくは少なくとも30個、例えば40個、60個または100個の連続した配列を一つまたは複数選択してプローブを設計し、例えばECL direct nucleic acid labeling and detection system(Amersham Biosciences社製)を用いて、マニュアルに記載の条件において、ハイブリダイズするポリヌクレオチドを指す。より具体的には、「ストリンジェントな条件」とは、例えば、通常、42℃、2×SSC、0.1% SDSの条件であり、好ましくは50℃、2×SSC、0.1% SDSの条件であり、さらに好ましくは、65℃、0.1×SSCおよび0.1% SDSの条件であるが、これらの条件に特に制限されない。ハイブリダイゼーションのストリンジェンシーに影響する要素としては、温度や塩濃度など複数の要素があり、当業者であればこれら要素を適宜選択することで最適なストリンジェンシーを実現することが可能である。 The enzyme that can be used in the present invention is also encoded by a polynucleotide that can hybridize under stringent conditions with a polynucleotide having a base sequence complementary to a polynucleotide containing the base sequence shown in SEQ ID NO: 1 or 3, for example. And an enzyme that satisfies the activity described above. The polynucleotide capable of hybridizing under stringent conditions is any at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in the nucleotide sequence set forth in SEQ ID NO: 1 or 3. One or a plurality of is selected, and a probe is designed. For example, using ECL direct nucleic acid labeling and detection system (manufactured by Amersham Biosciences), it refers to a polynucleotide that hybridizes under the conditions described in the manual. More specifically, “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions. More preferably, the conditions are 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.
本発明の方法で使用される植物油は、未精製の植物油(以下、「未精製油」ともいう)である。植物油としては、食用として使用され得る植物油が好ましく、例えば、大豆油、菜種油、ヒマワリ油、綿実油、紅花油、落花生油、米油などが挙げられる。未精製油は、粗油、または予備的に水脱ガムした油であり得る。水脱ガムは、粗油に水を添加してまたは粗油に酸性水溶液(好ましくはpH3〜6の酸性水溶液)を添加して、加温(例えば、30〜90℃)下で攪拌(通常、0.5〜9時間)することによって行われ得る。酸性水溶液として、有機酸またはリン酸あるいはそれらの塩が使用され得る。有機酸あるいは有機酸塩は、好ましくは、酢酸、クエン酸あるいはそれらの塩である。より好ましくは、酢酸、リン酸、クエン酸などを1〜100mM含みpHを3〜6の範囲に調整したものである。 The vegetable oil used in the method of the present invention is an unrefined vegetable oil (hereinafter also referred to as “unrefined oil”). As the vegetable oil, vegetable oil that can be used for food is preferable, and examples thereof include soybean oil, rapeseed oil, sunflower oil, cottonseed oil, safflower oil, peanut oil, and rice oil. The unrefined oil can be a crude oil or an oil that has been pre-water degummed. Water degumming is performed by adding water to a crude oil or adding an acidic aqueous solution (preferably an acidic aqueous solution having a pH of 3 to 6) to the crude oil, and stirring under heating (eg, 30 to 90 ° C.) (usually, 0.5-9 hours). An organic acid or phosphoric acid or a salt thereof can be used as the acidic aqueous solution. The organic acid or organic acid salt is preferably acetic acid, citric acid or a salt thereof. More preferably, it contains 1 to 100 mM of acetic acid, phosphoric acid, citric acid and the like, and the pH is adjusted to the range of 3-6.
植物油を水および酵素で処理する工程については、未精製油に対して添加された水および酵素が未精製油中のリン脂質(ガム質)に作用できる限り、水および酵素の添加の順序または様式は問わない。植物油を水および酵素で処理する工程は、通常の植物油の製造における「脱ガム工程」(すなわち、粗油からリン脂質を水和析出させてガム質として取り除く工程)の間に行うことができる。植物油を水および酵素で処理する工程としては、例えば、酵素を水もしくは適当な水溶液に分散溶解して添加するか、または、酵素および水もしくは適当な水溶液を個々に添加し、これらを油中に分散させ、ガム質を水和させる;予め粗油に水もしくは適当な水溶液を添加してガム質を予備的に水和させ、次いで酵素(好ましくは水と共に)を添加するなどが挙げられる。本明細書中において、植物油を水および酵素で処理する工程における酵素添加後の処理を単に「酵素処理」という。 For the process of treating vegetable oil with water and enzyme, the order or mode of addition of water and enzyme as long as the water and enzyme added to the unrefined oil can act on the phospholipids (gum) in the unrefined oil Does not matter. The step of treating the vegetable oil with water and an enzyme can be performed during a “degumming step” (that is, a step in which phospholipids are hydrated and precipitated from the crude oil and removed as a gum) in normal vegetable oil production. Examples of the process of treating vegetable oil with water and an enzyme include, for example, adding the enzyme by dispersing and dissolving the enzyme in water or an appropriate aqueous solution, or adding the enzyme and water or an appropriate aqueous solution individually, and adding these into the oil. Disperse and hydrate the gum; for example, water or a suitable aqueous solution is added to the crude oil in advance to pre-hydrate the gum and then an enzyme (preferably with water) is added. In the present specification, the treatment after addition of the enzyme in the step of treating vegetable oil with water and an enzyme is simply referred to as “enzyme treatment”.
酵素処理は、好ましくはpH3〜10、より好ましくはpH4〜9、さらにより好ましくはpH5〜9の範囲内で行う。上記pHになるように、酵素を添加する前、同時、もしくは後に、アルカリ(例えば、水酸化ナトリウム)が添加され得る。 The enzyme treatment is preferably performed within the range of pH 3 to 10, more preferably pH 4 to 9, and even more preferably pH 5 to 9. Alkaline (eg, sodium hydroxide) can be added before, simultaneously with, or after the enzyme is added to achieve the above pH.
酵素処理は、当該酵素の至適温度にもよるが、通常25〜80℃、好ましくは30〜60℃の条件下で、通常0.5〜8時間、好ましくは0.5〜5時間、行われ得る。 Although the enzyme treatment depends on the optimum temperature of the enzyme, it is usually 25 to 80 ° C., preferably 30 to 60 ° C., usually 0.5 to 8 hours, preferably 0.5 to 5 hours. Can be broken.
添加される酵素の量は、好ましくは、未精製油1kg当たり100〜5000ユニット、より好ましくは300〜3000ユニットである。添加される酵素の量はまた、好ましくは、酵素処理あたり1〜1000ppm、より好ましくは10〜500ppmである。 The amount of enzyme added is preferably 100 to 5000 units, more preferably 300 to 3000 units per kg of unrefined oil. The amount of enzyme added is also preferably 1-1000 ppm per enzyme treatment, more preferably 10-500 ppm.
酵素処理に添加される水の量は、好ましくは、未精製油100質量部当たり0.5〜10質量部、より好ましくは0.5〜7.5質量部、さらにより好ましくは0.5〜5質量部である。 The amount of water added to the enzyme treatment is preferably from 0.5 to 10 parts by weight, more preferably from 0.5 to 7.5 parts by weight, even more preferably from 0.5 to 100 parts by weight per 100 parts by weight of unrefined oil. 5 parts by mass.
「脱ガム工程」において添加される水の量は、植物油中のガム質を水和させるのに必要な量である限り、特に制限はないが、好ましくは、粗油100質量部に対して0.5〜10質量部、より好ましくは0.5〜7.5質量部、さらにより好ましくは0.5〜5質量部である。「脱ガム工程」において添加される水は、予備的な水脱ガムに用いる水、酵素処理に用いられる水の合計であり得る。 The amount of water added in the “degumming step” is not particularly limited as long as it is an amount necessary to hydrate the gum in the vegetable oil, but is preferably 0 with respect to 100 parts by mass of the crude oil. 5 to 10 parts by mass, more preferably 0.5 to 7.5 parts by mass, and even more preferably 0.5 to 5 parts by mass. The water added in the “degumming step” may be the total of water used for preliminary water degumming and water used for enzyme treatment.
植物油に添加される成分(例えば、酸性水溶液、アルカリ、酵素、および水)は、植物油に添加した後、適宜の乳化機(例えば、高速撹拌機、ホモミキサー、コロイドミル、パイプラインミキサー、超音波分散装置、高圧ホモジナイザー、バイブレーター、膜乳化装置など)を用いて乳化され得る。 Components added to the vegetable oil (eg, acidic aqueous solution, alkali, enzyme, and water) are added to the vegetable oil, and then an appropriate emulsifier (eg, high-speed stirrer, homomixer, colloid mill, pipeline mixer, ultrasonic wave) Emulsification using a dispersing device, a high-pressure homogenizer, a vibrator, a membrane emulsifying device, or the like).
酵素処理後、酵素を失活させるために加熱処理することが望ましい。 After the enzyme treatment, it is desirable to perform a heat treatment in order to deactivate the enzyme.
酵素処理は、バッチ式、多段バッチ式、あるいは連続式で行い得る。 The enzyme treatment can be performed in a batch system, a multistage batch system, or a continuous system.
植物油の精製方法では、酵素処理の後、ガム質が除去された植物油(すなわち、「脱ガム油」)を分離回収する。例えば、遠心分離機などの適宜手段によって、油相と水相とを分離し、分離後、油相を回収する。油相は、脱ガム油を含む。回収した油相をさらに上記の水脱ガムに供してもよい。 In the method for purifying vegetable oil, the vegetable oil from which the gum has been removed (ie, “degummed oil”) is separated and recovered after the enzyme treatment. For example, the oil phase and the aqueous phase are separated by appropriate means such as a centrifuge, and the oil phase is recovered after separation. The oil phase contains degummed oil. The recovered oil phase may be further subjected to the above water degumming.
脱ガム油は、常法による脱色工程(色素を活性白土などに吸着させて除去する)および脱臭工程(臭気化合物を蒸留除去する)にさらに供され得る。脱臭工程において、脂肪酸もまた揮発除去され得る。 The degummed oil can be further subjected to a decoloring step (removing the pigment by adsorbing it on activated clay) and a deodorizing step (distilling off the odorous compound) by a conventional method. In the deodorization step, fatty acids can also be volatilized off.
リゾリン脂質の製造方法では、酵素処理の後、リゾリン脂質を分離回収する。リゾリン脂質の分離回収のために、例えば、上述の水相を回収する。この水相をアセトン洗浄し、減圧乾燥することによって、リゾリン脂質が分離回収され得る。 In the method for producing lysophospholipid, lysophospholipid is separated and recovered after the enzyme treatment. For the separation and recovery of lysophospholipid, for example, the above-mentioned aqueous phase is recovered. By washing the aqueous phase with acetone and drying under reduced pressure, lysophospholipids can be separated and recovered.
また、リゾリン脂質の分離回収は、酵素処理後の液への水の添加によるガム質の水和析出;溶媒系(例えばn−ヘキサンと80(v/v)%エタノール水溶液)を用いる液々抽出;超臨界二酸化炭素による分別抽出などの公知の方法によっても行われ得る。 In addition, lysophospholipids are separated and recovered by hydration precipitation of gum by adding water to the solution after enzyme treatment; liquid-liquid extraction using a solvent system (for example, n-hexane and 80 (v / v)% ethanol aqueous solution) It can also be carried out by known methods such as fractional extraction with supercritical carbon dioxide.
分離回収したリゾリン脂質は、さらにシリカゲル、セルロースイオン交換体などの担体を用いた液体クロマトグラフィーなどの適宜手段によって精製することができる。 The separated and recovered lysophospholipid can be further purified by appropriate means such as liquid chromatography using a carrier such as silica gel or cellulose ion exchanger.
本発明では、上記のように植物油を精製しながら、該植物油からリゾリン脂質を製造し得る。 In the present invention, lysophospholipid can be produced from the vegetable oil while purifying the vegetable oil as described above.
以下、実施例により本発明をより具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these Examples.
(調製例1)
ストレプトマイセス・エバミチルスNBRC14893株からゲノムDNAを調製し、このゲノムDNAを鋳型として、配列番号5のフォワードプライマーおよび配列番号6のリバースプライマーを用いてPCR増幅し、遺伝子断片を得た。この遺伝子断片の塩基配列およびアミノ酸配列はそれぞれ、配列番号3および4に記載のとおりである。
(Preparation Example 1)
Genomic DNA was prepared from Streptomyces evarcylus NBRC14893 strain, and PCR amplification was performed using this genomic DNA as a template, using a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 to obtain a gene fragment. The base sequence and amino acid sequence of this gene fragment are as described in SEQ ID NOs: 3 and 4, respectively.
pTONA5(特開2009−65837号公報)を制限酵素NdeIおよびHindIIIで処理し、上記遺伝子断片をNdeIおよびHindIIIで処理し、得られた2つの断片をライゲーションした後、大腸菌JM109に導入した。カナマイシンを選択マーカーにして、遺伝子断片含有pTONA5プラスミドを得た。得られた遺伝子断片含有プラスミドを接合性大腸菌S17−1に導入し、その形質転換された大腸菌から接合形質転換によりストレプトマイセス・リビダンス(Streptomyces lividans)1326株を形質転換した。なお、接合形質転換法については、以下のように行った:
1)遺伝子断片含有pTONA5プラスミドを保持する接合大腸菌(S17−1)をカナマイシン50μg/mL入りLB培地2〜5mL中で37℃にて終夜振とう培養した;
2)培養液1mLを遠心分離(8,000rpm、5分間、4℃)後、上清をデカンテーションで捨てた;
3)菌体ペレットをLB培地1mLに懸濁した後、上記操作2)と同様にして菌体を洗浄した。この操作を2回繰り返した;
4)洗浄した菌体ペレットを500μLのLB培地を添加し、ピペットで穏やかに懸濁させた;
5)上記操作4で得た大腸菌懸濁液から100μLを分取し、放線菌ストレプトマイセス・リビダンス1326株の胞子懸濁液(1010個/mL)10μLとピペッティングで混合した;
6)ISP4培地平板培地(DifcoTM ISP Medium 4:3.7%)に混合液の全量を塗布し、30℃にて18時間培養した;そして
7)カナマイシン50μg/mLおよびナリジキシン酸ナトリウム67μg/mLを含むNBソフトアガー(DifcoTM Nutrient broth:0.8%、寒天:0.5%)3mLを重層し、30℃にて5日間培養して、形質転換株を得た。
pTONA5 (Japanese Patent Laid-Open No. 2009-65837) was treated with restriction enzymes NdeI and HindIII, the above gene fragment was treated with NdeI and HindIII, and the resulting two fragments were ligated and then introduced into E. coli JM109. A gene fragment-containing pTONA5 plasmid was obtained using kanamycin as a selection marker. The obtained gene fragment-containing plasmid was introduced into conjugative Escherichia coli S17-1, and Streptomyces lividans strain 1326 was transformed from the transformed Escherichia coli by conjugation transformation. The mating transformation method was performed as follows:
1) A conjugated Escherichia coli (S17-1) carrying a gene fragment-containing pTONA5 plasmid was cultured overnight at 37 ° C. in 2-5 mL of LB medium containing 50 μg / mL of kanamycin;
2) 1 mL of culture broth was centrifuged (8,000 rpm, 5 minutes, 4 ° C.), and the supernatant was discarded by decantation;
3) After the cell pellet was suspended in 1 mL of LB medium, the cell was washed in the same manner as in the above operation 2). This operation was repeated twice;
4) 500 μL of LB medium was added to the washed cell pellet and gently suspended with a pipette;
5) 100 μL of the Escherichia coli suspension obtained in the above operation 4 was collected and mixed by pipetting with 10 μL of the spore suspension (1010 cells / mL) of Streptomyces lividans strain 1326;
6) All the mixture was applied to ISP4 medium plate medium (Difco ™ ISP Medium 4: 3.7%) and incubated at 30 ° C. for 18 hours; and 7) Kanamycin 50 μg / mL and sodium nalidixate 67 μg / mL NB soft agar containing 3 mL of NB soft agar (Difco ™ Nutrient broth: 0.8%, agar: 0.5%) was overlaid and cultured at 30 ° C. for 5 days to obtain a transformant.
形質転換株を、グルコース2%、K2HPO4 0.8%、ポリペプトン(日本製薬製)0.5%、イーストエキストラクト(Difco製)0.5%、カナマイシン50μg/mL、pH7.0の培地において、28℃にて160rpmで72時間振盪培養した。培養培地は、500mL容バッフル付三角フラスコに25mLをそれぞれ分注し、121℃にて20分間の蒸気滅菌を予め行った(カナマイシンは滅菌後添加した)。菌体を除去した培養上清を酵素溶液として得た。 The transformed strain was glucose 2%, K 2 HPO 4 0.8%, polypeptone (manufactured by Nippon Pharmaceutical) 0.5%, yeast extract (Difco) 0.5%, kanamycin 50 μg / mL, pH 7.0. In the medium, shaking culture was performed at 28 ° C. and 160 rpm for 72 hours. The culture medium was dispensed in 25 mL into 500 mL baffled Erlenmeyer flasks, and steam sterilized at 121 ° C. for 20 minutes in advance (kanamycin was added after sterilization). The culture supernatant from which the cells were removed was obtained as an enzyme solution.
(酵素活性の測定)
以下の実施例および比較例でそれぞれ使用した酵素であるPLA2ナガセ(ナガセケムテックス株式会社製のホスホリパーゼA2)、調製例1の酵素、およびLecitase Ultra(非特許文献1)ならびに他のPLA2製品(Maxapal A2;DSM社製;ブタ膵臓由来PLA2コードポリペプチドをアスぺルギルス・ニガー(Aspergillus niger)で発現させた)について、ホスホリパーゼA活性、リゾホスホリパーゼ活性、リパーゼ活性、およびPLA2活性に対するPLA1活性を調べた:
ホスホリパーゼA活性:大豆レシチンを基質にpH8.0、37℃で反応した際に、1分間に1μmolの脂肪酸を遊離する活性を1ユニットとする;
リゾホスホリパーゼ活性:リゾホスファチジルコリン(Sigma, L0906)を基質にpH8.0、37℃で反応した際に、1分間に1μmolの脂肪酸を遊離する活性を1ユニットとする;
リパーゼ活性:リパーゼキットS(DSファーマバイオメディカル株式会社)を使用して、以下の手順で測定した。5,5’-ジチオビス(2-ニトロ安息香酸)を含む発色液1mL、検体50μLを混和後、フェニルメチルスルホニルフルオリドを含むエステラーゼ阻害液20μLを添加した。混和後、恒温槽で30℃にて5分間予熱した。予熱終了後、三酪酸ジメルカプロール、ドデシル硫酸ナトリウムを含む基質液100μLを加え、混和後30℃にて30分間インキュベーションした。反応停止液2mLを加え、反応を停止させた。吸光度412nmを測定した;
PLA2活性に対するPLA1活性:10%レシチン溶液(Ultralec P(ADM社)を水に分散)0.2mLに、酵素を300U/gとなるように添加し、37℃にて60分反応させた後、クロロホルム:メタノール(2:1(v/v))10mLを添加して、脂質を抽出し、遠心した上清を、HPLC法(カラム:Spherisorb Amino(Waters)(4.6μm, 250×150mm)、溶離液:エタノール(95%(v/v)):20mMシュウ酸=92:8(v/v)、流速1.0mL/分、カラム温度 室温、検出RI)に供し、1−アシルリゾリン脂質および2−アシルリゾリン脂質のピーク面積を求め、それぞれのリゾリン脂質を定量し、これらのそれぞれの定量値からPLA1活性とPLA2活性との比を算出することにより決定した。「ホスホリパーゼA2活性に対するホスホリパーゼA1活性」は、PLA2活性を100としたときのPLA1活性を%として表す。
(Measurement of enzyme activity)
PLA2 Nagase (phospholipase A2 manufactured by Nagase ChemteX Corporation), the enzyme of Preparation Example 1, Lecitase Ultra (Non-patent Document 1), and other PLA2 products (Maxapal), which are enzymes used in the following Examples and Comparative Examples, respectively. A2; manufactured by DSM; a porcine pancreas-derived PLA2-encoding polypeptide was expressed in Aspergillus niger), phospholipase A activity, lysophospholipase activity, lipase activity, and PLA1 activity against PLA2 activity were examined :
Phospholipase A activity: When reacting soy lecithin with a substrate at pH 8.0 and 37 ° C., the activity of releasing 1 μmol of fatty acid per minute is defined as 1 unit;
Lysophospholipase activity: When lysophosphatidylcholine (Sigma, L0906) is reacted with a substrate at pH 8.0 and 37 ° C., the activity of releasing 1 μmol of fatty acid per minute is defined as 1 unit;
Lipase activity: Using a lipase kit S (DS Pharma Biomedical Co., Ltd.), the lipase activity was measured by the following procedure. After mixing 1 mL of a coloring solution containing 5,5′-dithiobis (2-nitrobenzoic acid) and 50 μL of the sample, 20 μL of an esterase inhibitor containing phenylmethylsulfonyl fluoride was added. After mixing, it was preheated at 30 ° C. for 5 minutes in a thermostatic bath. After completion of preheating, 100 μL of a substrate solution containing dimercaprol tributyrate and sodium dodecyl sulfate was added, and the mixture was mixed and incubated at 30 ° C. for 30 minutes. 2 mL of a reaction stop solution was added to stop the reaction. Absorbance measured at 412 nm;
PLA1 activity with respect to PLA2 activity: Enzyme was added to 0.2 mL of 10% lecithin solution (Ultralec P (ADM) dispersed in water) at 300 U / g and reacted at 37 ° C. for 60 minutes, followed by chloroform. : 10 mL of methanol (2: 1 (v / v)) was added to extract the lipid, and the centrifuged supernatant was subjected to HPLC method (column: Spherisorb Amino (Waters) (4.6 μm, 250 × 150 mm), eluent) : Ethanol (95% (v / v)): 20 mM oxalic acid = 92: 8 (v / v), flow rate 1.0 mL / min, column temperature room temperature, detection RI), 1-acyl lysophospholipid and 2-acyl lysophospholipid The peak area was determined, each lysophospholipid was quantified, and the ratio between PLA1 activity and PLA2 activity was calculated from each quantified value. “Phospholipase A1 activity relative to phospholipase A2 activity” represents PLA1 activity as% when PLA2 activity is taken as 100.
上記測定により得られた活性に基づき、ホスホリパーゼA活性に対するリゾホスホリパーゼ活性(%)およびホスホリパーゼA活性に対するリパーゼ活性(%)を算出した。それぞれの酵素について、ホスホリパーゼA活性に対するリゾホスホリパーゼ活性(%)、ホスホリパーゼA活性に対するリパーゼ活性(%)、PLA2活性に対するPLA1活性の結果を以下の表1に示す。 Based on the activity obtained by the above measurement, lysophospholipase activity (%) for phospholipase A activity and lipase activity (%) for phospholipase A activity were calculated. Table 1 below shows the results of lysophospholipase activity (%) for phospholipase A activity, lipase activity (%) for phospholipase A activity, and PLA1 activity for PLA2 activity for each enzyme.
PLA2活性に対するPLA1活性は、表1では「PLA2/PLA1」および「%」で表す。例えば、PLA2/PLA1=100/0.2は、PLA2活性に対するPLA1活性が0.2%であることを表す。PLA2ナガセ、調製例1の酵素、およびMaxapal A2は、PLA2活性に対するPLA1活性が5%以下であり、PLA2活性を有する酵素であることが確認できた。Lecitase Ultraは、PLA2/PLA1=0/100であり、これは、PLA1活性のみが測定され、PLA2活性が全くみられなかったことを表す。 PLA1 activity relative to PLA2 activity is expressed in Table 1 as “PLA2 / PLA1” and “%”. For example, PLA2 / PLA1 = 100 / 0.2 indicates that PLA1 activity relative to PLA2 activity is 0.2%. PLA2 Nagase, the enzyme of Preparation Example 1, and Maxapal A2 had a PLA1 activity of 5% or less with respect to the PLA2 activity, and were confirmed to be enzymes having PLA2 activity. For Lecitase Ultra, PLA2 / PLA1 = 0/100, indicating that only PLA1 activity was measured and no PLA2 activity was observed.
PLA2ナガセ、調製例1の酵素、およびMaxapal A2は、ホスホリパーゼA活性に対するリゾホスホリパーゼ活性が5%以下であり、Lecitase Ultraは、6.4%であった。リゾホスホリパーゼ活性の割合が高くなれば、生成したリゾリン脂質が分解してしまう。 PLA2 Nagase, the enzyme of Preparation Example 1, and Maxapal A2 had a lysophospholipase activity of 5% or less with respect to phospholipase A activity, and Lecitase Ultra was 6.4%. If the ratio of lysophospholipase activity increases, the produced lysophospholipid will be degraded.
PLA2ナガセおよび調製例1の酵素はリパーゼ活性がほとんどなく、ホスホリパーゼA活性に対するリパーゼ活性が2%以下であるのに対し、Maxapal A2は、ホスホリパーゼA活性に対するリパーゼ活性が3.4%であり、Lecitase Ultraは、7115%であった。酵素処理では、処理対象である粗油中の主成分はトリグリセリド(約95%)であり、その粗油中に含まれる微量のリン脂質(約2%)を特異的に分解することが必要であり、そのためリパーゼ活性が少しでも副活性として含まれていると、粗油の主成分であるトリグリセリドに作用してしまい、精製油の収量が下がってしまう。 PLA2 Nagase and the enzyme of Preparation Example 1 have almost no lipase activity, and lipase activity with respect to phospholipase A activity is 2% or less, whereas Maxapal A2 has lipase activity with respect to phospholipase A activity of 3.4%. Ultra was 7115%. In the enzyme treatment, the main component in the crude oil to be treated is triglyceride (about 95%), and it is necessary to specifically decompose a small amount of phospholipid (about 2%) contained in the crude oil. Therefore, if the lipase activity is contained as a side activity even a little, it acts on the triglyceride which is the main component of the crude oil, and the yield of the refined oil is lowered.
(精製油およびリゾリン脂質の評価方法)
以下の実施例および比較例で用いた測定法は以下の通りである:
リンの定量には、ICP発光分析法を用いた;
トリグリセリドの定量には、HPLC法(カラム:Inertsil SIL 100A (5μm, 250×150mm)、溶離液:ヘキサン/2−プロパノール(996/4)、流速1.0mL/分、カラム温度 40℃、検出RI)を用いた;
リゾレシチンに含まれるリゾリン脂質の種類の決定は、リゾレシチンをクロロホルム/メタノール混合溶液(2/1(v/v))に溶解して、上記PLA2活性に対するPLA1活性の決定に用いたのと同じHPLC法に供し、ピーク位置から1−アシルリゾリン脂質または2−アシルリゾリン脂質を決定した。
(Method for evaluating refined oil and lysophospholipid)
The measurement methods used in the following examples and comparative examples are as follows:
ICP emission spectrometry was used for the quantification of phosphorus;
For quantification of triglycerides, HPLC method (column: Inertsil SIL 100A (5 μm, 250 × 150 mm), eluent: hexane / 2-propanol (996/4), flow rate 1.0 mL / min, column temperature 40 ° C., detection RI) Was used;
Determination of the type of lysophospholipid contained in lysolecithin is the same HPLC method used to determine PLA1 activity relative to PLA2 activity by dissolving lysolecithin in a chloroform / methanol mixed solution (2/1 (v / v)). The 1-acyl lysophospholipid or 2-acyl lysophospholipid was determined from the peak position.
(実施例1)
100gの大豆粗油(リン含量650ppm)を50℃に加温し、次いでこれに50質量%クエン酸水溶液0.13mLを添加し、これをホモミキサーを用いて13,000rpmにて1分間処理し、その後、50℃にて1時間緩やかに撹拌した。これに4N NaOH 315μLを添加し、これをホモミキサーを用いて13,000rpmにて1分間処理し、次いでこれにPLA2ナガセを30ppm添加し、さらに水を添加して系内の水相の量を2.5mLに調整した。これをホモミキサーを用いて13,000rpmにて1分間処理し、その後、37℃にて2時間緩やかに撹拌した。80℃にて30分間加熱して酵素を失活させた後、3,500rpmにて10分間遠心分離し、上層の油相(脱ガム油)および下層の水相をそれぞれ回収した。
Example 1
100 g soybean crude oil (phosphorus content 650 ppm) is heated to 50 ° C., and then 0.13 mL of 50 mass% citric acid aqueous solution is added thereto, and this is treated at 13,000 rpm for 1 minute using a homomixer, and then The mixture was gently stirred at 50 ° C. for 1 hour. To this was added 315 μL of 4N NaOH, and this was treated at 13,000 rpm for 1 minute using a homomixer. Then, 30 ppm of PLA2 Nagase was added thereto, and water was further added to adjust the amount of the aqueous phase in the system to 2.5. Adjusted to mL. This was treated at 13,000 rpm for 1 minute using a homomixer, and then gently stirred at 37 ° C. for 2 hours. After inactivating the enzyme by heating at 80 ° C. for 30 minutes, the mixture was centrifuged at 3,500 rpm for 10 minutes to recover the upper oil phase (degummed oil) and the lower aqueous phase, respectively.
油相中のリン量を定量した。また、油相中のトリグリセリド量を定量し、事前に定量していた粗油中のトリグリセリド量を用いてトリグリセリドの収率を算出した。 The amount of phosphorus in the oil phase was quantified. Further, the amount of triglyceride in the oil phase was quantified, and the yield of triglyceride was calculated using the amount of triglyceride in crude oil that had been quantified in advance.
水相をアセトンで洗浄した後、減圧乾燥し、リゾレシチンを得た。リゾレシチン量を定量し、リゾレシチンに含まれるリゾリン脂質の種類を決定した。 The aqueous phase was washed with acetone and then dried under reduced pressure to obtain lysolecithin. The amount of lysolecithin was quantified, and the type of lysophospholipid contained in lysolecithin was determined.
(実施例2)
37℃にて2時間の撹拌条件を50℃にて2時間に変更した以外は、実施例1と同様の手順を行った。
(Example 2)
The same procedure as in Example 1 was performed except that the stirring condition for 2 hours at 37 ° C. was changed to 2 hours at 50 ° C.
(実施例3)
37℃にて2時間の撹拌条件を50℃にて2時間に変更し、そしてPLA2ナガセの添加量を30ppmから300ppmに変更した以外は、実施例1と同様の手順を行った。
(Example 3)
The same procedure as in Example 1 was performed, except that the stirring conditions for 2 hours at 37 ° C were changed to 2 hours at 50 ° C, and the addition amount of PLA2 Nagase was changed from 30 ppm to 300 ppm.
(実施例4)
PLA2ナガセを調製例1の酵素に変更し、その添加量を36Uに変更した以外は、実施例1と同様の手順を行った。
Example 4
The same procedure as in Example 1 was performed except that PLA2 Nagase was changed to the enzyme of Preparation Example 1 and the addition amount was changed to 36 U.
(比較例1)
PLA2ナガセをLecitase Ultraに変更し、4N NaOHの添加量を315μLから110μLに変更した以外は、実施例1と同様の手順を行った。
(Comparative Example 1)
The same procedure as in Example 1 was performed except that PLA2 Nagase was changed to Lecitase Ultra and the amount of 4N NaOH added was changed from 315 μL to 110 μL.
(比較例2)
37℃にて2時間の撹拌条件を50℃にて2時間に変更した以外は、比較例1と同様の手順を行った。
(Comparative Example 2)
The same procedure as in Comparative Example 1 was performed except that the stirring conditions for 2 hours at 37 ° C were changed to 2 hours at 50 ° C.
(比較例3)
37℃にて2時間の撹拌条件を50℃にて2時間に変更し、そしてLecitase Ultraの添加量を30ppmから300ppmに変更した以外は、比較例1と同様の手順を行った。
(Comparative Example 3)
The same procedure as in Comparative Example 1 was performed except that the stirring condition for 2 hours at 37 ° C was changed to 2 hours at 50 ° C, and the addition amount of Lecitase Ultra was changed from 30 ppm to 300 ppm.
実施例1〜4および比較例1〜3について、油相(脱ガム油)中のリン量、トリグリセリドの収率、得られたリゾレシチン量およびリゾレシチンに含まれるリゾリン脂質の種類を以下の表2に示す。 For Examples 1 to 4 and Comparative Examples 1 to 3, the amount of phosphorus in the oil phase (degummed oil), the yield of triglycerides, the amount of lysolecithin obtained and the types of lysophospholipids contained in lysolecithin are shown in Table 2 below. Show.
表2の結果から、PLA2ナガセを用いた実施例1〜3によれば、脱ガム油中のリン含量は全て25ppm未満であり、十分に精製されていることが分かった。実施例1〜3のいずれにおいてもトリグリセリド収率は約95%以上であり、37℃および50℃の両反応とも優れていた。調製例1の酵素を用いた実施例4によっても、脱ガム油中のリン含量は25ppm未満であり、十分に精製され、そして96%という高いトリグリセリド収率が得られた。 From the results in Table 2, it was found that according to Examples 1 to 3 using PLA2 Nagase, the phosphorus contents in the degummed oil were all less than 25 ppm and were sufficiently refined. In any of Examples 1 to 3, the triglyceride yield was about 95% or more, and both the reactions at 37 ° C. and 50 ° C. were excellent. Also according to Example 4 using the enzyme of Preparation Example 1, the phosphorus content in the degummed oil was less than 25 ppm, fully purified, and a high triglyceride yield of 96% was obtained.
Lecitase Ultraを用いた比較例では、脱ガム油中のリン含量は全て25ppm未満であり、十分に精製されていたが、トリグリセリド収率に関しては、比較例2の50℃反応では、約95%の高い収率が得られたが、比較例1の37℃反応ではトリグリセリドが分解し、結果としてトリグリセリドの収率は低かった。50℃反応であっても酵素量を増大した比較例3では、リン脂質以外のトリグリセリドも分解してしまい、トリグリセリドの収率は低かった。 In the comparative example using Lecitase Ultra, the phosphorus content in the degummed oil was all less than 25 ppm and was sufficiently refined, but the triglyceride yield was about 95% in the 50 ° C. reaction of Comparative Example 2. Although a high yield was obtained, triglyceride was decomposed in the 37 ° C. reaction of Comparative Example 1, and as a result, the yield of triglyceride was low. In Comparative Example 3 in which the amount of enzyme was increased even at 50 ° C., triglycerides other than phospholipids were also decomposed, and the yield of triglycerides was low.
Lecitase Ultraはリパーゼ活性を含む(表1)ため、37℃程度の温度ではリパーゼ活性によりトリグリセリドを分解する。リパーゼ活性を抑制するためには、高温(例えば50℃)にする必要があり、これは、エネルギー効率的に好ましくない。高温下であっても酵素量を増大させると、リパーゼ活性によりトリグリセリドを分解する。一般的に粗油に含有されるリン脂質にはロット差があるので、酵素の添加量を粗油ロットごとに調節する必要が生じ、望ましくない。 Since Lecitase Ultra contains lipase activity (Table 1), triglycerides are degraded by lipase activity at a temperature of about 37 ° C. In order to suppress lipase activity, it is necessary to make it high temperature (for example, 50 degreeC), and this is not preferable in terms of energy efficiency. When the amount of the enzyme is increased even at high temperature, the triglyceride is decomposed by the lipase activity. Generally, since there are lot differences in phospholipids contained in crude oil, it is necessary to adjust the amount of enzyme added for each crude oil lot, which is not desirable.
実施例1〜4で得られたリゾリン脂質は1−アシルリゾリン脂質であり、これは、保存安定性が高い。他方、比較例1〜3で得られたリゾリン脂質は2−アシルリゾリン脂質であり、これは、保存安定性が低く分解されやすい。 The lysophospholipids obtained in Examples 1 to 4 are 1-acyl lysophospholipids, which have high storage stability. On the other hand, the lysophospholipid obtained in Comparative Examples 1 to 3 is a 2-acyl lysophospholipid, which has low storage stability and is easily degraded.
さらに、比較例3では、リゾレシチン量が低くなり、得られたリゾリン脂質の純度が下がった。これは、Lecitase Ultraのリゾホスホリパーゼ活性によって、生成したリゾリン脂質がさらに分解されたことによる。 Furthermore, in Comparative Example 3, the amount of lysolecithin was reduced, and the purity of the obtained lysophospholipid was lowered. This is because the produced lysophospholipid was further degraded by the lysophospholipase activity of Lecitase Ultra.
本発明によれば、植物油を効率的に精製できる方法、およびリゾリン脂質を効率的に製造できる方法が提供される。さらに、本発明によれば、植物油を精製すると共に、リゾリン脂質もまた製造できる方法が提供される。よって、本発明は、植物油の製造、ならびに食品および化粧品用乳化剤として有用なリゾリン脂質の製造に役立つ。 According to the present invention, there are provided a method capable of efficiently purifying vegetable oil and a method capable of efficiently producing lysophospholipid. Furthermore, the present invention provides a method for purifying vegetable oil and producing lysophospholipids. Thus, the present invention is useful for the production of vegetable oils and lysophospholipids useful as food and cosmetic emulsifiers.
Claims (4)
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、および
該処理後の植物油から脱ガム油を分離回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素が、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である、
方法。 A method for refining vegetable oil,
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity, and separating and recovering degummed oil from the treated vegetable oil,
The enzyme having phospholipase A2 activity is
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
Method.
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、および
該処理後の植物油からリゾリン脂質を分離回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素が、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である、
方法。 A method for producing lysophospholipids from vegetable oil,
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity, and separating and recovering lysophospholipids from the treated vegetable oil,
The enzyme having phospholipase A2 activity is
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
Method.
該植物油を、水およびホスホリパーゼA2活性を有する酵素で処理する工程、および
該処理後の植物油を油相および水相に分離する工程、
該油相を回収する工程、および
該水相からリゾリン脂質を回収する工程
を含み、
該ホスホリパーゼA2活性を有する酵素が、
ホスホリパーゼA2活性に対するホスホリパーゼA1活性が5%以下であり、そして
ホスホリパーゼA活性に対し、リゾホスホリパーゼ活性が5%以下、かつ、リパーゼ活性が2%以下である、
方法。 A method for refining vegetable oil and producing lysophospholipids from the vegetable oil,
Treating the vegetable oil with water and an enzyme having phospholipase A2 activity, and separating the treated vegetable oil into an oil phase and an aqueous phase;
Recovering the oil phase, and recovering lysophospholipid from the aqueous phase,
The enzyme having phospholipase A2 activity is
The phospholipase A1 activity is 5% or less with respect to the phospholipase A2 activity, and the lysophospholipase activity is 5% or less and the lipase activity is 2% or less with respect to the phospholipase A activity.
Method.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20160138552A (en) * | 2014-04-01 | 2016-12-05 | 피피지 인더스트리즈 오하이오 인코포레이티드 | Electrode binder composition for lithium ion electrical storage devices |
JP2018085984A (en) * | 2016-11-18 | 2018-06-07 | 株式会社明治 | Enzyme-treated sweetened egg yolk, and pudding using the same |
JP7297135B1 (en) | 2022-10-06 | 2023-06-23 | キユーピー株式会社 | Method for producing lysophospholipid-containing composition, and method for producing oil-in-water emulsion composition using the same |
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2012
- 2012-06-06 JP JP2012128617A patent/JP2013252077A/en active Pending
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20160138552A (en) * | 2014-04-01 | 2016-12-05 | 피피지 인더스트리즈 오하이오 인코포레이티드 | Electrode binder composition for lithium ion electrical storage devices |
KR102023238B1 (en) | 2014-04-01 | 2019-09-19 | 피피지 인더스트리즈 오하이오 인코포레이티드 | Electrode binder composition for lithium ion electrical storage devices |
JP2018085984A (en) * | 2016-11-18 | 2018-06-07 | 株式会社明治 | Enzyme-treated sweetened egg yolk, and pudding using the same |
JP7297135B1 (en) | 2022-10-06 | 2023-06-23 | キユーピー株式会社 | Method for producing lysophospholipid-containing composition, and method for producing oil-in-water emulsion composition using the same |
WO2024075335A1 (en) * | 2022-10-06 | 2024-04-11 | キユーピー株式会社 | Method for producing lysophospholipid-containing composition and method for producing oil-in-water emulsion composition using same |
JP2024054894A (en) * | 2022-10-06 | 2024-04-18 | キユーピー株式会社 | Method for producing lysophospholipid-containing composition, and method for producing oil-in-water type emulsion composition using the same |
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